(1) Field of the Invention
The present invention relates generally to plasmid vectors, and more particularly to Actinobacillus succinogenes plasmid vectors. The present invention further relates to overexpression of proteins in A. succinogenes and to engineered A. succinogenes strains which have the plasmids introduced into them.
(2) Description of the Related Art
Succinate has many industrial fine chemical uses. Succinate can also be used as an intermediary commodity chemical feedstock for producing bulk chemicals. Its greatest market potential might lie in its use as feedstock to produce stronger-than-steel plastics, biodegradable chelators, and green solvents. Most of the 17,000 tons (15,400 metric tons) of succinate sold per year are produced petrochemically from maleic acid. Succinate is also produced as a fine chemical by fermentation from glucose. For fermentation to be competitive in producing succinate as a commercial chemical, the overall production cost should be lowered from one (1) dollar per pound ($2.20 per kg) to approximately twenty (20) cents per pound (44 cents per kg).
U.S. Pat. No. 5,143,833 to Datta et al. teaches a method for producing succinic acid by growing a succinate producing Anaerobiospirillum succiniciproducens microorganism under specific conditions.
U.S. Reissued Patent No. RE37,393 to Donnelly et al. teaches a method for isolating succinic acid producing bacteria by increasing the biomass of an organism which lacks the ability to catabolize pyruvate and then growing the biomass in glucose-rich medium under an anaerobic environment to enable pyruvate-catabolizing mutants to grow. By using this method, Donnelly provides a mutant E. coli that produces high amounts of succinic acid. The mutant E. coli was derived from a parent which lacked the genes for pyruvate-formate lyase and lactate dehydrogenase.
U.S. Pat. No. 6,455,284 and U.S. Pat. Application Publication No. 2003/0087381, both to Gokarn et al. teach metabolic engineering to increase the carbon flow toward oxaloacetate to enhance production of bulk biochemicals, such as lysine and succinate, in bacterial and industrial fermentations. The carbon flow is redirected by genetically engineering bacteria to overexpress the enzyme pyruvate carboxylase.
U.S. Pat. Application Publication No. 2003/0113885 to Lee et al. teaches a novel microorganism, Mannheimia sp. 55E, capable of producing organic acids and a process using the microorganism for producing organic acids through anaerobic and aerobic incubations.
U.S. Pat. No. 6,420,151 to Eikmanns, et al. teaches an isolated nucleic acid from coryneform bacteria which encodes a phosphoenolpyruvate carboxykinase which is involved in production of succinate.
While the above methods can be used to produce succinate, Actinobacillus succinogenes is still the best succinate producer known. Actinobacillus succinogenes is a gram-negative capnophilic, anaerobic bacillus that belongs to Pasteurellaceae. A. succinogenes produces up to one hundred (100) grams per liter of succinate in optimized conditions. Much effort has been spent on engineering Escherichia coli strains to produce high succinate amounts, however none of the engineered E. coli strains surpassed A. succinogenes for succinate production. Carbon flow is stringently regulated in microorganism metabolism, including carbon flux towards oxaloacetate. Overcoming this control of carbon flux will possibly improve the yields of desirable products, such as succinate.
Previously, no genetic tools had been tested or developed that could be used for engineering A. succinogenes into an industrial succinate-producing strain. Therefore, it would be desirable to have a means to genetically engineer A. succinogenes to overproduce succinate. However, there is not yet a means for constructing recombinant A. succinogenes. The present invention provides a means for constructing recombinant A. succinogenes using a plasmid for the expression of proteins in A. succinogenes. 